Dietary long-chain fatty acids (LCFAs) contribute up to 40% of the caloric content in western diets. LCFAs are important metabolites and contribute to many cellular structures and functions, but excessive serum fatty acid levels are linked to obesity, insulin desensitization, and type 2 diabetes. LCFA uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). In humans, six members of this transporter family, designated as hsFATP1-6, have been characterized and shown to be differentially expressed throughout the body. The FATP4 subtype constitutes the major FATP in the small intestine and preferentially uptakes long- (>10 carbon atoms) but not short-chain fatty acids. Selective inhibition of this subtype FATP may effectively reduce LCFA absorption. High-throughput screening (HTS) of a large compound library will be the initial step toward identifying novel subtype-selective inhibitors of FATPs. Current methods using radiolabeled fatty acids or fluorescent fatty acid analogs to measure LCFA uptake in murine 3T3-L1 adipocytes are not suitable for HTS to identify subtype-selective inhibitors against human FATPs. In response to RAF-RM-06-004: "Assay Development for High Throughput Molecular Screening," we propose to develop and configure an HTS platform using a fluorometric imaging plate reader (FLIPR[unreadable]) with the newly developed QBT" Fatty Acid Uptake Assay Kit to quality LCFA uptake kinetics by the stable hsFATP4-expressing HEK293 cells. Our specific aims are to: 1. Develop a FLIPR 96- or 384-well format assay against the hsFATP4 for primary HTS. LCFA uptake kinetics in hsFATP4-expressing HEK293 cells will be compared with the vector control HEK293 cells and 3T3-L1 adipocytes to determine signal-to-noise ratios, specificity, and reproducibility for the HTS assay. Through assay optimization, we will configure the assay for primary HTS. 2. Evaluate and validate the HTSassay through an initial screening. A panel of several hundred compounds consisting of natural long- and short-chain fatty acids, synthetic fatty acid derivatives, and small molecules will be screened against the hsFATP4 as an initial evaluation. The HTS assay we develop could be used in the Molecular Library Screening Center Network for primary screens and will serve as a general prototype for subsequent development of assays for other FATP family members. Secondary screens will use the standard radiolabeled method against the same target for hit confirmation and the FLIPR assay against another FATP subtype in counter-screening for selectivity. The identified subtype-selective FATP inhibitors will provide a novel pharmacologic tool for further studies of FATP biology. Furthermore, they may form a basis for developing novel therapeutics for the treatment of metabolic disorders.